We are interested in elucidating the molecular mechanisms involved in tissue stem cell self-renewal and fate choice, in normal tissue development and homeostasis. Stem cells hold great promises for future therapies of numerous deadly diseases, yet their basic properties are not well understood. Progress in the field has been hampered by the difficulty of identifying tissue stem cells in the absence of specific biochemical markers. Increasing evidence suggested that stem cells are infrequently dividing cells, that could be identified in many mouse tissues as DMA label retaining cells (or LRCs). Recently we developed a strategy applicable to many self-regenerating tissues to pulse-label LRCs with the green fluorescent protein (GFP) fused to histone H2B. We have used the mouse skin and the hair follicle as model systems, since the relation between LRCs and skin epithelial stem cells had been investigated in most depth. In our preliminary work we tracked and isolated GFP LRCs and demonstrated that at least a fraction of these cells had a stem cell phenotype. In this proposal we describe in three Specific Aims strategies to examine in more detail the relation between LRCs and stem cells. We will: 1) classify LRCs into distinct sub- populations and investigate their sternness; 2) examine their special cell cycle properties; 3) characterize their distinct patterns of gene expression. To accomplish these goals we use in vivo cell tracking and in vitro cell sorting methods combined with a variety of assays which include: hair regeneration and skin wounding; cell culture and skin reconstitution; cell cycle kinetics; and microarrays. First, our data will have global relevance for the isolation and characterization of putative stem cells with LRCs properties from many tissues. Although we focus on the skin, our work will lead the way for similar approaches in many systems which lack the specific means to separate stem cells from other cells of their tissue of residence. Second, stem cell proliferation must be tightly controlled in tissues to regulate the balance between normal and abnormal growth. Our studies will unravel some of the mechanisms developed by putative SCs to control their proliferation, an important first step in understanding such perturbation in the diseased tissues. Finally, our work will provide some of the basic knowledge necessary for more efficient manipulation of skin stem cells for clinical applications such as cell transplantation on burn victims.